ProjectSummary:MetabolicandGeneticHeterogeneityinCancer Targetingglucosemetabolismincancerisanattractivetherapeuticarea,buteffortstodevelopsuccessful glycolytic inhibitors have failed. A major barrier in the cancer metabolism field is a systems-level understanding of the differential regulation of glycolysis that results from metabolic rewiring. This limitationpromptedtheinvestigationtodeterminewhethermetaboliccontrolcanbeexploitedfortherapy. ThehypothesisoftheF99phaseisthatrationaltargetingofpivotalenzymesthatdifferentiallyregulate centralcarbonmetabolismincancercanresultinanti-tumorefficacyandcharacterizationofresistance mechanismstoglycolysisinhibitioncanadvancethedevelopmentofcombinationtherapy.Toaddress thishypothesis,Aim1characterizestheroleoftwoimportantenzymesincentralcarbonmetabolism,3- phosphoglyceratedehydrogenase(PHGDH)andglyceraldehyde-3-phosphatedehydrogenase(GAPDH) andhowtheseenzymescanbetargetedforcancertherapy.Sub-aim1.1usesCRISPR/Cas9mediated PHGDHknockout(KO)tovalidatetheselectivityandactivityoftwonovelallostericPHGDHinhibitors. Sub-aim1.2usesquantitativedeterminantsoftheWarburgEffect(WE)torevealGAPDHasarational therapeutictarget,andcomparativemetabolomicstonominateKAasapotentGAPDHinhibitorinhighly glycolyticcells,togetheruncoveringmetabolicpredictorsofdrugresponsetoGAPDHinhibition.These findingscontributetoashiftingparadigminthecurrentunderstandingofmetaboliccancertherapyand show the potential use of metabolic predictors, rather than genetic determinants, to predict response. Aim 2 will seek to characterize the resistance mechanisms to GAPDH inhibition in order to develop combinationtherapyandbetterunderstandtherequirementsoftheWE.KA-sensitivecellswillbemade resistant and a lentiviral CRISPR/Cas9 sgRNA library loss-of-function screen will be employed to determine the metabolic enzymes driving resistance. The outcome will result in characterization of resistancedevelopmenttoglucosemetabolisminhibitionandthemechanismbywhichtheWEoccurs. Inaddition,sincetumorheterogeneitylargelycontributestolimitationsindrugtargeting,thereisanurgent need to better characterize the molecular determinants of microenvironment formation. The interplay amongenvironmentalfactors,genetics,andepigeneticshaverecentlybeenappreciated.Thehypothesis oftheK00phaseisthatadynamiccross-talkexistsbetweenoncogenicandepigeneticnetworksandis governedbytissueorigin.Aim3willexaminetheroleofoncogenicdriversandepigeneticregulatorsat different stages of cancer progression in different microenvironment settings to unravel the molecular componentscontributingtotumorheterogeneity.Theoutcomewillestablishabetterunderstandingof therelationshipbetweentumorheterogeneityandtherapeuticresponse.